The yeast vacuole resembles an animal cell lysosome, for it is an acidic compartment, contains a complement of hydrolases, and is the final destination of ligands taken up by fluid phase and receptor-mediated endocytosis. Since yeast is amenable to genetical, biochemical and molecular analysis and since fundamental biological processes have been almost obsessively conserved across eukaryotes, yeast offers an excellent model system for studies of the function and assembly of this important organelle. These studies may contribute to our understanding of the mannose-6-phosphate independent pathway of lysosomal/vacuolar enzyme targeting and of membrane assembly. They may enable understanding of the missorting of lysosomal proteases that occurs in some pathologic conditions like breast cancer. The overall goals of this research are to understand in molecular detail how proteins are targeted to the vacuole, how the organelle is assembled, and how it functions as a digestive and homeostatic organelle. Mutants unable to properly sort vacuolar hydrolases (pep7 and pep12) or to acidify (vph1) or assemble (pep5) the vacuole were isolated and the corresponding genes cloned. The specific aims in this proposal are 1) to determine whether Pepl2p, which is required for lumenal hydrolase targeting, is in both Golgi and vacuolar membranes, whether it functions by cycling between the two organelles, whether this cycling is essential for its function, what its function is, and what other proteins it interacts with as it carries out its functions 2) to determine whether Pep7p, which is required for lumenal hydrolase targeting, is in the nucleus, whether it functions as a transcription factor, what genes it regulates if it does and what the functions of the regulated genes are 3) to determine whether VPH1 and STV1 encode organelle specific ATPase subunits, what the organelles are, whether there are other members of the gene family, and if so, clone and study them, and to determine the topology of Vph1p in the vacuolar membrane 4) to determine the domain structure of Pep5p, which is required for formation of vacuoles, to identify other proteins with which it interacts in assembling the vacuole, and determine whether loss of function of these proteins gives similar phenotypes 5) to recover the gene(s) encoding the vacuolar Ca2+ transporter, make mutations, and use them to probe the role of the transporter and organelle in cellular Ca2+ homeostasis. Methods will include targeted mutagenesis, suppressor selection and analysis, subcellular fractionation, biochemistry, and immunofluorescence.